1. Cochlear involvement in tinnitus HELMY (WHAM) MULDERS FACULTY OF SCIENCE

2. The University of Western Australia Tinnitus affects 5-15% of population. Can severely affect quality of life No cure yet Strong correlation with hearing loss - Prevalence increasing Tinnitus What is the neural substrate of tinnitus? Perception-abnormal neuronal activity (Axelsson and Ringdahl, 1989)

3. The University of Western Australia 1. Changes in tonotopic maps Changes in the brain after hearing loss: Possible mechanisms for tinnitus 2. Synchronous activity between central structures 3. Increased spontaneous activity (hyperactivity) in central auditory pathway Note: several theories suggest these changes in the auditory system are accompanied/modulated by changes in non-auditory parts of the brain. Eggermont and Komiya, 2000; Robertson and Irvine 1989; Norena and Eggermont, 2003

4. The University of Western Australia Adapted from Lee Kishnan and Winer Recovery 0-12 weeks Record single neuron activity in inferior colliculus Behavioural tinnitus test Our guinea pig model to study central hyperactivity and tinnitus Pure tone acoustic trauma Record cochlear neural thresholds (CAP) to establish hearing loss

5. The University of Western Australia Our animal model to study tinnitus: Our results: Increased neural activity in IC without sound present: HYPERACTIVITY Mulders and Robertson Neurosci. 2010 ; Mulders et al. J. Neurosci. 2010 Hyperactivity High activity Low activity Control data

6. The University of Western Australia Hyperactivity shows correlation with region of hearing loss Robertson et al 2013 hearing Res Human studies: Audiogram vs tinnitus pitch: frequencies of hearing loss closely match pitch of perceived tinnitus

7. The University of Western Australia Measuring tinnitus in animals Normal animals Tinnitus animals Startle response Startle Background noise gap prepulse inhibition of the acoustic startle (GPIAS) gap Turner et al. 2006

8. The University of Western Australia Our guinea pig model: central hyperactivity and tinnitus Mulders et al., 2011 J. Comp. Neurol.; Robertson et al 2013 Hearing. Res Correlation hearing loss and hyperactivity Tinnitus: GPIAS Before acoustic trauma After acoustic trauma

9. The University of Western Australia Can we modulate the central hyperactivity? http://www.neuroreille.com/promenade/english/audiometry/ex_ptw/explo_ptw.htm Acoustic trauma Measure single neuron activity Hyperactivity x Stop activity auditory nerve

10. The University of Western Australia 1-6 weeks recovery: Acute destruction of auditory nerve after recovery period eliminates hyperactivity After acute cochlear ablationPost recovery-pre-ablation High activity Low activity Pitch increase Mulders and Robertson. 2009 Neuroscience

11. The University of Western Australia 8-12 weeks recovery: Acute destruction of auditory nerve does NOT completely eliminate hyperactivity Post-recovery-pre-ablationAfter acute cochlear ablation Mulders and Robertson 2011 Neurosci

12. The University of Western Australia Mulders and Robertson 2011 Neurosci

13. The University of Western Australia Central Hyperactivity-a two stage process? A therapeutic window?

14. The University of Western Australia The Big Question In stage 1: Reduction spontaneous activity in auditory nerve: hyperactivity In stage 1: Reduction spontaneous activity in auditory nerve: tinnitus ???

15. The University of Western Australia Possibility: Furosemide Loop diuretic (affecting membrane transport). Known to affect kidney and inner ear Decreases spontaneous firing rate auditory nerve fibres (Sewell 1984) Can suppress tinnitus in human subjects (Risey et al 1995; Caesarani et al. 2002) How can we suppress spontaneous activity of the auditory nerve fibres?

16. The University of Western Australia Adapted from Lee Kishnan and Winer Recovery 0-12 weeks Measure brain activity: effect of furosemide Behavioural tinnitus test: effect of furosemide Can we modulate hyperactivity and tinnitus in our animal model using furosemide? Partial deafness Measure hearing loss

17. The University of Western Australia N=4 Furosemide acutely decreases spontaneous firing auditory afferent nerve fibres (SNN) and central hyperactivity Furosemide (80 mg/kg i.p.) Mulders, Barry and Robertson 2014 Plos One

18. The University of Western Australia Saline i.p. has no effect on behavioural signs of tinnitus * ** SALINE Mulders, Barry and Robertson 2014 Plos One

19. The University of Western Australia Furosemide i.p. eliminates behavioural signs of tinnitus *** ** FUROSEMIDE SALINE Mulders, Barry and Robertson 2014 Plos One

20. The University of Western Australia In conclusion  Our data suggest that furosemide can suppress the behavioural signs of tinnitus in our animal model.  Our data strengthens the argument that hyperactivity is involved in the generation of tinnitus.  Our data supports the notion that there may be a therapeutic window for some time after acoustic trauma.

21. The University of Western Australia What’s next?  Can we show proof of principle in human tinnitus sufferers? (collaboration Prof Friedland; Ear Science Institute Australia)  Investigations into more chronic effects of furosemide on tinnitus. (Mulders et al. 2014 Frontiers in Neuroscience)  Other options beside furosemide?  What about treatments for centralized tinnitus?

22. The University of Western Australia A different way to modulate activity in the cochlea: Extra-cochlear electrical stimulation (ECES) Tested in our animal model using round window electrical stimulation Partial deafness Measure hearing loss ECES with positive current can suppress activity of auditory nerve Suppression of tinnitus reported using ECES with positive direct current Mechanism unknown-due to reduction of central hyperactivity? RW stimulation Schreiner et al. 1986; Cazals et al., 1978

23. The University of Western Australia Norena, Mulders and Robertson J. Neurophysiology Only small effect on thresholds and tone-induced activity of IC neurons. ECES may be a viable approach for suppressing some forms of (peripheral-dependent) tinnitus. ECES with negative current hyperactivity in IC ECES with positive current hyperactivity in IC

24. The University of Western Australia Acknowledgements: Action on Hearing Loss (UK) Neurotrauma Research Program NHMRC MHRIF Auditory lab members A/Prof. Jenny Rodger (University of Western Australia) Dr Arnaud Norena (Universite de Marseille, France) Prof Tony Paolini (RMIT, Australia) Prof Richard Salvi (University of Buffalo, USA)

25. The University of Western Australia Cochlear perfusion with kainic acid or CoCl 2 suppresses spontaneous activity 1-6 weeks recovery: Acute but temporary silencing of auditory nerve after recovery period eliminates hyperactivity Single neuron recordings in midbrain 2 weeks after acoustic trauma: Mulders et al. 2009 Neurosci

26. The University of Western Australia What’s next?  Can we show proof of principle in human tinnitus sufferers? (collaboration ESIA)  Investigations into more chronic effects of furosemide on tinnitus.  Other options beside furosemide?  What about treatments for centralized tinnitus?

27. The University of Western Australia Repetitive Transcranial Magnetic Stimulation (rTMS) Therapeutic effects on many neurological and psychiatric disorders Non-invasive Some success reported in tinnitus patients Vooys 2014; Huerta and Volpe, 2009; (Khedr et al., 2008, Langguth et al., 2008, Khedr et al., 2010

28. The University of Western Australia rTMS frequency protocol Low Frequency rTMS – inhibits neuronal activity High frequency rTMS – excites neuronal activity Hallet (2000); Huerta and Volpe (2009); Hoffman (2002); Eichhammer et al. (2003)

29. The University of Western Australia Can rTMS suppress hyperactivity after hearing loss? 10 Guinea Pigs Vooys 2014

30. The University of Western Australia Coil size and position 10minute sessions, 1 Hz, Monday – Friday for 2 weeks Vooys 2014 In preparation

31. The University of Western Australia rTMS does not affect hearing loss but reduces hyperactivity Vooys 2014 ***

32. The University of Western Australia Can rTMS reduce the behavioural signs of tinnitus in our animal model?

33. The University of Western Australia Preliminary data: possible effect rTMS on tinnitus? N=3/group Is rTMS affecting descending pathways from the cortex? Direct effect on IC?

34. The University of Western Australia Modulation of hyperactivity by paraflocculus (Darryl Vogler) Modulation of hyperactivity and tinnitus by limbic system (Kristin Barry and Prof Tony Paolini RMIT University Melbourne) Projection patterns of descending auditory systems (Ahmaed Bashaar) Effects of cochlear electrical stimulation on hyperactivity and tinnitus Testing validity of GPIAS in human subjects (Prof. Geoff Hammond, ESIA) Effects of rTMS on hyperactivity and tinnitus (A/Prof. Jenny Rodger) Proof of principle experiment effect of furosemide in tinnitus subjects (ESIA and Prof Peter Friedland) Further ongoing studies

35. The University of Western Australia Neural substrates of tinnitus Human studies: Neuroimaging data: Excessive spontaneous activity in auditory structures Audiogram vs tinnitus pitch: frequencies of hearing loss closely match pitch of perceived tinnitus Animal studies: (models of hearing loss) Electrophysiology: Increased spontaneous activity in auditory structures Audiogram vs tinnitus pitch: Increased spontaneous activity associated with frequency range of hearing loss and behavioral signs of tinnitus correlate with Increased spontaneous activity/ frequency range of hearing loss

36. The University of Western Australia RESULTS (STUDY2): SPONTANEOUS FIRING RATE

37. The University of Western Australia RESULTS (STUDY1): BDNF ELISA

38. The University of Western Australia Measuring brain activity Depth CF (characteristic frequency) and threshold Spontaneous firing rate 90-120 neurons per animal Picture courtesy C. Bester

39. The University of Western Australia Experiment 1: Guinea pig model Establish hyperactivity in the brain Measure excitatory and inhibitory systems in the brain: Gene products and brain chemicals Main result: Inhibitory systems Dong et al. Neurosci. 2009 Dong et al. Eur. J. Neurosci. 2010 Dong et al. Brain Res. 2010